Antibodies and pharmaceutical compositions containing same useful for inhibiting activity of metalloproteins

ABSTRACT

A method of producing a metalloprotein inhibitor, the method comprising generating antibodies directed at a composition including a metal ion-bound chelator, wherein the composition is selected having structural and electronic properties similar to a functional domain of the metalloprotein, thereby producing the metalloprotein inhibitor.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/175,978 filed on Jul. 5, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/382,248 filed on Mar. 11, 2009, now abandoned,which is a division of U.S. patent application Ser. No. 10/551,715 filedon Jul. 20, 2006, now U.S. Pat. No. 7,524,938, which is a National Phaseof PCT Patent Application No. PCT/IL2004/000308 filed on Apr. 4, 2004,which claims the benefit of priority of U.S. Provisional PatentApplication No. 60/460,005 filed on Apr. 4, 2003. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to antibodies and fragments thereof whichcan be used to inhibit activity of metalloproteins, such asmetalloproteases, and to methods which utilize same for treatingdiseases such as metastatic cancer which are associated with abnormalactivity of a metalloprotein.

The matrix metalloproteins (MMPs) are key enzymes participating inremodeling of the extracellular matrix (ECM). These enzymes are capableof destroying a variety of connective tissue components of articularcartilage or basement membranes.

The human MMP gene family consists of at least 28 structurally relatedproteins (see FIG. 1), which share a similar overall spherical topology(FIG. 2 and Borkakoti, 1998). Each MMP is secreted as an inactive,latent pro-enzyme. The catalytic zinc domain is composed of about 180amino acids wherein the highly conserved sequence HE-GH-LGL-H providesthe three histidine (i.e., H) residues which bind to the metal Zn(2+)ion. The forth-binding site of the catalytic zinc ion in the pro-enzymeis bound to a cystein residue (Morgunova et al., 1999), which uponenzyme activation dissociates from the active site (Van Wart andBirkedal-Hansen, 1990). As a result, the forth-binding site in theactivated MMPs is taken up by a water molecule, which is alsohydrogen-bonded to a conserved glutamic residue. This processfacilitates the hydrolysis of a peptide bond of the target substratewith the activated water molecule.

The uncontrolled breakdown of connective tissue by metalloproteases is afeature of many pathological conditions, probably resulting from anexcess of MMP activity or from an imbalanced ratio between the naturalMMP tissue inhibitors (TIMPs) and MMPs. TIMPs inhibit MMPs by formingstoichiometric complexes with the active zinc binding site of MMPs(Gomez et al., 1997; Henriet at al., 1999; Bode et al., 1999; Will etal., 1996). When TIMPs levels are insufficient, a progressive slowdegradation of the ECM may lead to loss of cartilage matrix inrheumatoid arthritis (Walakovits et al., Arthritis Rheum, 35:35-42,1992) and osteoarthritis (Dean et al., J. Clin. Invest. 84:678-685,1989) or bone matrix degradation in osteoporosis (Hill et al., Biochem.J. 308: 167-175, 1995). In other situations, such as congestive heartfailure, rapid degradation of the heart's ECM may occur (Armstrong etal., Canadian J. Cardiol. 10: 214-220, 1994).

Other pathological condition, which are also related to unregulatedactivity of MMPs, include the rapid remodeling of the ECM by metastatictumor cells. In such conditions the activated MMPs are either expressedby the cancer cells or by the surrounding tissues. There is considerableevidence that MMPs are involved in the growth and spread of tumors(e.g., see Davidson et al., Chemistry & Industry, 258-261, 1997, andreferences therein). In the process of tumor metastasis, MMPs are usedto break down the ECM, allowing primary tumor cancer cells to invadeneighboring blood vessels where they are transported to different organsand establish secondary tumors. The invasive growth at these secondarysites is mediated by MMPs, which break down the tissue. In addition, MMPactivity contributes to the invasive in-growth of new blood vessels,also termed angiogenesis, which is required for tumors to grow above acertain size.

Given the broad role of MMPs in human physiology and pathology, it isnot surprising that numerous efforts have been affected to design drugs,which inhibit MMP excessive activity.

Drug discovery efforts have focused on inhibitor classes that contain afunctional group which coordinates the zinc ion to thereby inactivatethe target MMP. One such inhibitor class is the hydroxamate inhibitors,small peptide analogs of fibrillar collagens, which specificallyinteract in a bidentate manner via the hydroxyl and carbonyl oxygens ofthe hydroxamic group with the zinc ion in the catalytic site [Grams etal., (1995), Biochem. 34: 14012-14020; Bode et al., (1994), EMBO J., 13:1263-1269].

Hydroxamate-based MMP inhibitors are usually composed of either a carbonback-bone (WO 95/29892, WO 97/24117, WO 97/49679 and EP 0780386), apeptidyl back-bone (WO 90/05719, WO 93/20047, WO 95/09841 and WO96/06074) or a peptidomimetic back-bone [Schwartz et al., Progr. Med.Chem., 29: 271-334(1992); Rasmussen et al., Pharmacol. Ther., 75: 69-75(1997); Denis et al., Invest. New Drugs, 15: 175-185 (1997)].Alternatively, they contain a sulfonamido sulfonyl group which is bondedon one side to a phenyl ring and a sulfonamido nitrogen which is bondedto an hydroxamate group via a chain of one to four carbon atoms (EP0757984 A1).

Other peptide-based MMP inhibitors are thiol amides which exhibitcollagenase inhibition activity (U.S. Pat. No. 4,595,700),N-carboxyalkyl derivatives containing a biphenylethylglycine whichinhibit MMP-3, MMP-2 and collagenase (Durette, et al., WO-9529689),lactam derivatives which inhibit MMPs, TNF-alpha and aggrecanase (seeU.S. Pat. No. 6,495,699) and Tricyclic sulfonamide compounds (see U.S.Pat. No. 6,492,422).

Although peptide-based MMP inhibitors have a clear therapeutic potentialtheir use in clinical therapy is limited. Peptide-based hydroxamate arecostly to produce and have low metabolic stability and oralbioavailability [e.g., batimastat (BB-94)]. These compounds are rapidlyglucuronidated, oxidized to carboxylic acid and excreted in the bile[Singh et al., Bioorg. Med. Chem. Lett. 5: 337-342, 1995; Hodgson,“Remodelling MMPIs”, Biotechnology 13: 554-557, 1995)]. In addition,peptide-based MMP inhibitors often exhibit the same or similarinhibitory effects against each of the MMP enzymes. For example,batimastat is reported to exhibit IC₅₀ values of about 1 to about 20 nMagainst each of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 [Rasmussen et al.,Pharmacol. Ther., 75(1): 69-75 (1997)]. Furthermore, the use of severalhydroxamate inhibitors was associated with severe side effects such asmuscoloskeletal problems with marimastat (BB-2516), widespreadmaculopapular rash with CGS27023A (Novartis) [Levitt et al., 2001, Clin.Cancer Res. 7: 1912-1922] and liver abnormalities, anemia, shoulder andback pain, thrombocytopenia, nausea, fatigue, diarrhea and deep veinthrombosis with BAY12-9566 (Bayer) [Heath et al., 2001, CancerChemother. Pharmacol. 48: 269-274]. Moreover, phase III clinical trialson advanced cancer patients with marimastat, prinomastat (AG 3340,Agouron) and Bay 12-9566 demonstrated no clinical efficacy in inhibitingmetastasis (Zucker et al., 2000, Oncogene 19: 6642-50).

Other MMP inhibitors are the chemically modified nonmicrobialtetracyclines (CMTs) that were shown to block expression of several MMPsin vitro. However, in vivo efficacy of these compounds was found to belimited, e.g., the CMT inhibitor, doxycycline, reduced tissue levels ofMMP-1 but not MMP-2, 3, or -9 in atherosclerotic carotid plaques inhuman patients (Axisa et al., 2002, Stroke 33: 2858-2864).

Recently, a mechanism-based MMP inhibitor, SB-3CT, was designedaccording to the X-ray crystallographic information of the MMP activesite (Brown et al., 2000). X-ray absorption studies revealed thatbinding of this molecule to the catalytic zinc reconstructs theconformational environment around the active site metal ion back to thatof the pro-enzyme [Kleifeld et al., 2001, J Biol. Chem. 276: 17125-31].However, the therapeutic efficacy obtained with this agent is yet to bedetermined.

Another class of natural inhibitors is monoclonal antibodies. Severalantibodies have been raised against specific peptide sequences withinthe catalytic domain MMP-1 (Galvez et al., 2001, J. Biol. Chem., 276:37491-37500). However, although these antibodies could inhibit thein-vitro activity of MMP, results demonstrating the in-vivoeffectiveness of such antibodies have not been demonstrated.

As described hereinabove, the catalytic site of MMPs includes acoordinated metal ion which becomes available for substrate bindingfollowing enzyme activation (see FIGS. 2 a-c). It is thus conceivablethat conventional antibodies directed at the primary amino acid sequenceof the enzyme would not distinguish the active form from the inactiveform of the enzyme and hence would not serve as potent inhibitors ofsuch enzymes.

While reducing the present invention to practice, the present inventorshave uncovered that in sharp contrast to the above, antibodies whichrecognize electronic and structural determinants of the catalytic siteof MMPs are potent inhibitors thereof and as such can be used to treatdiseases associated with imbalanced MMP activity.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anantibody or an antibody fragment, comprising an antigen recognitionregion capable of binding a metal ion and a chelator thereof, whereinthe antibody or the antibody fragment is capable of inhibiting anactivity of a metalloprotein.

According to another aspect of the present invention there is provided amethod of producing a metalloprotein inhibitor, the method comprisinggenerating antibodies directed at a composition including a metalion-bound chelator, wherein the composition is selected havingstructural and electronic properties similar to a functional domain ofthe metalloprotein, thereby producing the metalloprotein inhibitor.

According to yet another aspect of the present invention there isprovided an antibody or an antibody fragment, comprising an antigenrecognition region capable of binding a metal ion and a chelatorthereof, wherein the antibody or the antibody fragment is capable ofinhibiting an activity of a matrix metalloprotease.

According to still another aspect of the present invention there isprovided a pharmaceutical composition comprising an antibody or anantibody fragment including an antigen recognition region capable ofbinding a metal ion and a chelator thereof and a physiologicallyacceptable carrier, wherein the antibody or antibody fragment is capableof inhibiting an activity of a matrix metalloprotease.

According to an additional aspect of the present invention there isprovided a matrix metalloprotease inhibitor comprising an antibody or anantibody fragment including an antigen recognition region capable ofbinding a metal ion and a chelator thereof.

According to yet an additional aspect of the present invention there isprovided a method of producing a matrix metalloprotease inhibitor, themethod comprising generating antibodies directed at a compositionincluding a metal ion-bound chelator, wherein the composition isselected having structural and electronic properties similar to acatalytic domain of the matrix metalloprotease, thereby producing thematrix metalloprotease inhibitor.

According to still an additional aspect of the present invention thereis provided a method of inhibiting matrix metalloprotease activity in asubject in need thereof, the method comprising providing to the subjecta therapeutically effective amount of an antibody or an antibodyfragment including an antigen recognition region capable of binding ametal ion and a chelator thereof, thereby inhibiting matrixmetalloprotease activity in the subject.

According to a further aspect of the present invention there is providedan article-of-manufacture comprising packaging material and apharmaceutical composition identified for treating diseases associatedwith abnormal activity of a matrix metalloprotease being containedwithin the packaging material, the pharmaceutical composition including,as an active ingredient, an antibody or an antibody fragment includingan antigen recognition region capable of binding a metal ion and achelator thereof, wherein the antibody or antibody fragment is capableof inhibiting an activity of the matrix metalloprotease.

According to further features in preferred embodiments of the inventiondescribed below, the metal ion is a transition metal ion selected fromthe group consisting of Vanadium, Selenium, Molybdenum, Cobalt, Zinc,Copper, Iron, Gallium, Bismuth, Aluminum, Gold, Platinum, Manganese,Chronium, Silver, Antimony, Thalium, Cadmium, Nickel, Mercury and Lead.

According to still further features in the described preferredembodiments the chelator is a polyamine.

According to still further features in the described preferredembodiments the polyamine is at least two histidine molecules.

According to still further features in the described preferredembodiments the polyamine is selected from the group consisting ofethylene diamine, cyclam, porphyrin, diethylenetriamine,triethylenetetramine, triethylenediamine, tetraethylenepentamine,aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine,captopril, penicilamine, N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-Bis-(2-animoethyl)-1,3-propanediamine,1,7-dioxa-4,10-diazacyclododecane, 1,4,8,11-tetraazacyclotetradecane-5,7-dione, 1,4,7-triazacyclononane,1-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and1,4,7,10-tetraazacyclododecane.

According to still further features in the described preferredembodiments the metalloprotein is selected from the group consisting ofneutrophil collagenase, collagenase-3, gelatinase A, gelatinase B,stromelysins-2 and 3, matrilysin, macrophage elastase; membrane-typeMMPs, agrrecanase, tumor necrosis factor converting enzyme, cytokineconvertases, adhesion molecule shedding enzymes, endothelin convertingenzyme, angiotensin converting enzyme, neutral endopeptidase,FTSH—bacterial metalloprotease, metallo-lactamase (carbapenases),bacterial toxins and ras farnesyl protein transferase and carbonicanhydrase.

According to yet a further aspect of the present invention there isprovided a method of qualifying specificity of an antibody to a metalion and a chelator thereof, the method comprising determiningconformational changes in binding of the metal ion to the chelatorthereof following binding of the antibody, to thereby qualify thespecificity of the antibody to the metal ion and the chelator thereof.

According to still further features in the described preferredembodiments the antibodies are polyclonal antibodies.

According to still further features in the described preferredembodiments the antibodies are monoclonal antibodies.

According to still a further aspect of the present invention there isprovided a method of qualifying specificity of an antibody to a metalion and a chelator thereof, the method comprising determining electronicchanges in the metal ion following binding of the antibody, to therebyqualify the specificity of the antibody to the metal ion and thechelator thereof.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing metalloprotein antibodies,such as MMP antibodies, which can be used to treat diseases associatedwith abnormal MMP activity.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic illustration of the MMP protein family domainstructure as adapted from Nagase et al., 1999. Shown are the signalpeptide, pro-peptide, catalytic, fibronectin type II, linker andhemopexin-like domains. The various groups of domain organization(numbered A through H) represent the structure of the following MMPproteins: A: Matrilysin (MMP-7); B: Collagenase 1 (MMP-1), Stromelysin 1(MMP-3), Collagenase 2 (MMP-8), Stromelysin 2 (MMP-10), Macrophageelastase (MMP-12), Collagenase 3 (MMP-13), Collagenase 4 (Xenopus,MMP-18), MMP-19, Enamelysin (MMP-20), CMMP (chicken, MMP-22); C:Gelatinase A (MMP-2); D: Gelatinase B (MMP-9); E: Stromelysin 3(MMP-11); F: MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT3 (MMP-16), MT4(MMP-17); G: XMMP (Xenopus, MMP-21); and H: MMP-23.

FIG. 2 a is a schematic illustration depicting the 3D structure ofproMMP-2 as adapted from the protein data bank (PDB): 1CFK(http://www.rcsb.org/pdb) demonstrating the proMMP-2 structural domains:the pro-domain (red), the catalytic domain (blue), the fibronectin typeII domains 1-3 (green) and the hemopexin domain (yellow).

FIG. 2 b is a schematic illustration of a structural alignment of thecatalytic sites in MMP-2, MMP-9, and TACE.

FIG. 2 c is a schematic illustration of the structural alignment of FIG.2 b zooming on the metal binding domain.

FIG. 3 is a schematic illustration depicting the molecular structure ofCo/Zn-TCPP hapten wherein “M” represents Co or Zn.

FIG. 4 is a titration curve of the purified Co-TCPP monoclonal antibody.Serial dilutions of the Co-TCPP monoclonal antibody were added totetra-carboxy phenyl porphyrin Co(II) (Co-TCPP) hapten-coatedmicro-titer plates. The absorbance of bound antibodies was measured at280 nm and was plotted against log concentration of the antibody.

FIG. 5 is a graphic illustration depicting a competitive inhibitionassay of the purified Co-TCPP monoclonal antibody binding to theindicated haptens. The antibody was incubated (at IC₅₀) with Co-TCPP(purple), TCPP (blue), Zn-TCPP (yellow), Zn-TPP (light blue) haptens atthe indicated concentrations and the relative fraction of boundantibodies (% binding) was calculated.

FIGS. 6 a-b are Lineweaver-Burk plots of MMP-2 and MMP-9 depicting thehydrolysis of a fluorogenic substrate in the presence of the Co-TCPPmonoclonal antibody. The MMP-2 catalytic domain (FIG. 6 a) and the MMP-9full length (FIG. 6 b) proteins were incubated with increasingconcentrations of the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ fluorogenicsubstrate in the presence of 6 μM (FIGS. 6 a-b, purple squares), 18 μM(FIGS. 6 a-b, yellow triangles) 24 μM (FIGS. 6 a-b, light blue Xs), orabsence (FIGS. 6 a-b, blue diamonds) of the antibody.

FIG. 7 a illustrates an immunoprecipitation (IP) assay of MMP-2 with theCo-TCPP monoclonal antibody. An SDS-PAGE gel loaded with the soluble orpellet phases obtained from the following IP experiments is shown;Incubation of MMP-2 with protein A beads (FIG. 7 a, Lane 1: soluble,Lane 2: pellet); incubation of the antibody with protein A beads (Lane3: soluble, Lane 4: pellet); incubation of MMP-2 with pre-associatedantibody-protein A beads (Lane 5: soluble, Lane 6: pellet); incubationof Dead Box Proten A (DbpA) with pre-associated antibody-protein A beads(Lane 7: soluble, Lane 8: pellet) and incubation of MMP-2 with antibody(Lane 9: soluble). Molecular weight marker (1 μg) is shown in Lane 10.Bands were visualized using Coomassie Blue staining.

FIG. 7 b illustrates an immunoprecipitation assay of a zinc-free MMP-2.Shown is an SDS-PAGE of the soluble (Lane 1) or pellet (Lane 2) phasesobtained by incubating the zinc-free MMP-2 with pre-associatedantibody-protein A beads. Bands were visualized using Coomassie bluestaining.

FIG. 7 c illustrates an immunoprecipitation assay of MMP-2 in thepresence or absence of the GM-6001 MMP inhibitor. Shown is an SDS-PAGEof the pellet or soluble phases obtained by the incubation of MMP-2 withpre-associated antibody-protein A beads (pellet phase: Lane 1), or bythe incubation of MMP-2 with pre-associated antibody-protein A beads inthe presence of GM-6001 (soluble phase: Lane 2, pellet phase: Lane 3).Molecular weight marker (1 μg) is shown in Lane 4. Bands were visualizedusing Coomassie Blue staining.

FIGS. 8 a-b illustrate the Zinc K-edge spectra of MMP-2. Shown are thenormalized X-ray absorption spectroscopy (XAS) data of the full-lengthlatent (FIGS. 8 a-b, green), active (FIGS. 8 a-b, black) and inhibited(FIGS. 8 a-b, red) forms of MMP-2. Note the small peak at 9680 eV (FIG.8 a, arrow 1) and the absence of cleft at 9730 eV (FIG. 8 a, arrow 2) inthe inhibited enzyme. Also note the distinct shift of 0.86 electronvolts (eV) to higher energy between the active and inhibited enzymes(FIG. 8 b).

FIG. 9 illustrates an extended X-ray absorption fine structure (EXAFS)analysis of the monoclonal antibody-Zinc immunocomplex. Results arepresented in the R-space of the experimental data (black) to simulatedtheoretical zinc ligand contributions (red).

FIG. 10 is an SDS PAGE analysis demonstrating 4-aminophenylmercuricacetate (APMA) binding to MMP-2 immunocomplexes. Recombinant MMP-2catalytic domain was incubated with pre-associated antibody-protein Abeads in the presence or absence of the MMP activator, APMA. Shown is anSDS-PAGE of the soluble or pellet phases obtained following theincubation of MMP-2 with pre-associated antibody-protein A beads (Lane1, pellet phase) or incubation of MMP-2 with pre-associatedantibody-protein A beads in the presence of APMA (FIG. 10, lane 2:soluble, lane 3: pellet). Bands were visualized with Coomassie bluestaining.

FIGS. 11 a-b are photomicrographs depicting the ability of the mAb ofthe present invention to block pericellular proteolysis generated byhighly invasive fibrocarcoma cancer cells (HT1080), as determined bysitu zymography assay. FIG. 11 a shows pericellular proteolytic activityof MMP-expres sing HT1080 cells (in light blue; cell nucleui are stainedwith DAPI). FIG. 11 b shows pericellular proteolytic activity of HT1080cells in the presence of mAb (1 μg). Note, MMP-mediated pericellularproteolysis which appears as green intensity around the cell membrane(FIG. 11 a) disappears upon incubation of the cells with the mAb (FIG.11 b).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of antibodies and fragments thereof, which canbe used to inhibit metalloprotein activity. Specifically, the antibodiesof the present invention can be used to treat diseases associated withimbalanced matrix metalloprotease activity such as metastatic cancers.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Matrix metalloproteases participate in many biological processes,ranging from cell proliferation, differentiation and remodeling of theextracellular matrix (ECM) to vascularization and cell migration. Theseprocesses require a delicate balance between the functions of the matrixmetalloproteases (MMPs) and natural tissue inhibitors thereof (TIMPs).The loss of this balance is the hallmark of numerous pathologicalconditions including metastatic tumors, neurodegenerative diseases andosteoarthritis.

Numerous MMP inhibitors are known in the art including small peptideinhibitors such as hydroxomate, non-microbial tetracyclins andmonoclonal antibodies. While the former are limited by the high cost ofproduction, high degradability, low oral bioavailability and lack ofspecificity, none of the latter have demonstrated in-vivo therapeuticefficacy.

While reducing the present invention to practice and while searching fora novel therapeutic modality to clinical conditions associated withimbalanced metalloenzyme activity, the present inventors have uncoveredthat antibodies which recognize both electronic and structuraldeterminants of the catalytic site of metalloenzymes can be used aspotent inhibitors thereof.

These findings enable, for the first time, to generate highly efficienttherapeutic antibodies which can be used to treat clinical conditionscharacterized by elevated metalloprotein activity.

Thus, according to one aspect of the present invention, there isprovided a method of producing a metalloprotein inhibitor.

The method is effected by generating antibodies or antibody fragmentsdirected at a composition which includes a metal ion-bound chelator.Such a composition is selected having structural and electronicproperties similar to a functional domain, such as a catalytic domain ora substrate binding domain, of the metalloprotein.

As used herein a “metalloprotein” refers to a protein, which includes abound metal ion as part of a structure thereof. The metal ion may berequired for enzymatic activity (i.e., metalloenzyme), eitherparticipating directly in catalysis, or stabilizing the activeconformation of the protein.

It will be appreciated that all members of the MMP family are translatedas latent enzymes, which upon activation are converted into activeenzymes in which the metal ion in the active site is accessible forsubstrate binding. For example, the “cysteine switch model” has beenpreviously suggested to explain MMP in vitro activation. The cysteineswitch model suggests that upon activation, the latent zinc-binding siteis converted to a catalytic zinc-binding site by dissociation of thethiol (Cys)-bearing propeptide from the zinc atom. Cleavage of thepropeptide results in a breakdown of the pro-domain structure of theenzyme, and the shielding of the catalytic zinc ion is withdrawn.Consequently, the metal ion and the active site pocket are accessiblefor substrate binding and hydrolysis [Van Wart and Birkedal-Hansen(1990) Proc. Natl. Acad. Sci. USA 87, 5578-5582].

Unlike prior art antibodies, the antibodies and antibody fragments ofthis aspect of the present invention serve as potent inhibitors of MMPs,due to their ability to bind both the metal ion and the coordinatingamino acids within the catalytic zinc site, thereby specificallyinhibiting the active conformation of these enzymes which are directlyinvolved in pathological processes as described above.

Examples of metalloenzymes which may be inhibited using the teachings ofthe present invention, include but are not limited to, neutrophilcollagenase, collagenase-3, gelatinase A, gelatinase B, stromelysins-2and 3, matrilysin, macrophage elastase; membrane-type MMPs, agrrecanase,cytokine convertases, adhesion molecule “shedding enzymes”, endothelinconverting enzyme, angiotensin converting enzyme, neutral endopeptidase,FTSH—bacterial metalloprotease, metallo-lactamase (carbapenases),bacterial toxins e.g., tetanus or botulism toxins, ras farnesyl proteintransferase, carbonic anhydrase and the like. Other examples ofmetalloenzymes are disclosed in Hodgson, Bio/Technology, 13:554 (1995);Gordon, et al., Clin. Exper. Rheum., 11(8):S91-S94 (1993); Ray, et al.,Eur. Respir. J., 7:2062-2072 (1994); O'Connor, et al., Thorax, 49:602-609 (1994); Docherty, et al., Tibtech, Vol. 10, (1992); Newby, etal., Basic Res. Cardiol., 89(Suppl):59-70; Freije, et al., J. Biol.Chem., 269(24):16766-16773 (1994); Shapiro, et al., J. Biol. Chem.,268(32):23824-23829 (1993); Belaauoaj, et al., J. Biol. Chem.,27(24):14568-14575 (1995); Gearing, et al., Letters to Nature, Nature,370:555-557 (1994); McGeehan, et al., Letters to Nature, Nature,370:558-561 (1994); Mohler, et al., Letters to Nature, Nature,370:218-220 (1994); Sato, et al., Letters to Nature, Nature, 370:61-65(1994); Crowe, et al., J. Exp. Med., 181:1205-1210 (1995); Payne, J.Med. Microbiol., 32:93-99 (1993); Deshpande, et al., Toxicon,33(4):551-557 (1995); DePhillips, et al., Eur. J. Biochem., 229:61-69(1995).

As described hereinabove any composition including a metal ion bound toa chelator thereof can be used to generate the antibodies of the presentinvention, as long as it has structural and electronic (i.e., charge,e.g., charge distribution and/or density) properties similar to that ofa functional domain of the metalloprotease. Although use of the actualmetal ion-bound chelator domain of the metalloprotein in generatingantibodies is preferred, it will be appreciated that in cases whereinsuch a domain is well characterized, one may synthesize and utilizestructures which mimic the structural and electronic properties of thisdomain, methods of synthesizing such metal ion-bound chelators aredescribed in detail hereinbelow.

As used herein the metal ion refers to a transition metal ion or anyother physiological metal. Examples of such metal ions include but arenot limited to Vanadium, Selenium, Molybdenum, Cobalt, Zinc, Copper,Iron, Gallium, Bismuth, Aluminum, Gold, Platinum, Manganese, Chronium,Silver, Antimony, Thalium, Cadmium, Nickel, Mercury, Lead, Magnesium,and Calcium.

It will be appreciated that although each of the transition metal ionsdescribed above can be included in the immunizing compositions of thisaspect of the present invention, a preferred metal ion is thatcoordinated within the chelating sequence of the natural metalloprotein.For example, since natural MMPs enclose Zinc at the catalytic domain, apreferred metal ion according to this aspect of the present invention isZinc or its analogous ions Cobalt or Cademium.

As used herein, the term “chelator” refers to a transition metalchelator, which includes least two atoms capable of coordinating anindicated metal to form a ring.

As is well known in the art, one or more molecules are considered astransition metal chelators if the formation of a cyclic complex of themolecule(s) with an ion of the transition metal results in a “chelateeffect”. The phrase “chelate effect” refers to the enhanced stability ofa complexed system containing the chelate, as compared with thestability of a system that is as similar as possible but contains noneor fewer rings. The parameters for evaluating the chelate effect of achelate typically include the enthalpy and entropy changes (ΔH and ΔS),according the following equation:

ΔG ⁰ =ΔH ⁰ −TΔS ⁰ =−RT ln β

where β is the equilibrium constant of the chelate formation and hencerepresents the chelate effect. Transition metal chelates refer tocomplexes, which include a transition ion and one or more transition ionchelator(s) complexed therewith, which are characterized by a large βvalue.

Examples of transition metal chelators include, but are not limited to,polyamine molecules such as porphyrins, ethylene diamine and cyclam,which form metal chelates with enhanced chelate effect.

Other examples of metal chelators include diethylenetriamine,di-(monoalkylamino)-alkane, triethylenetetramine, triethylenediamine,tetraethylenepentamine, aminoethylethanolamine, aminoethylpiperazine,pentaethylenehexamine, captopril, penicilamine, N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-Bis-(2-animoethyl)-1,3-propanediamine,1,7-dioxa-4,10-diazacyclododecane, 1,4,8,11-tetraazacyclotetradecane-5,7-dione, 1,4,7-triazacyclononane,1-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and1,4,7,10-tetraazacyclododecane. For further discussion on metalchelators by Ross and Frant, Chelometric indicators, titration with thesolid state cupric ion selective electrode. (1969) Analytical Chemistry41:1900.

It will be appreciated that peptide chelators can also be used accordingto this aspect of the present invention. U.S. Pat. No. 5,679,548discloses a method of generating such chelators.

In any case as described above the chelating composition is selectedbased upon the structural and electronic properties of the actual domainin the target polypeptide. Typically, the target polypeptide includes 3amino acids which provide three contact points required for thetransition metal coordination. Representative coordination complexgeometries can be tetrahedral, square planar or trigonal depending uponthe transition metal ion. In general the mimicking compositions of thepresent invention are selected based upon the the amino acid side chainstructure and the geometry of coordination. Typically, amino acids whichcan coordinate transition metal binding are histidine, arginine,glutamate, cysteine, methionine, tryptophan, serine, threonine andtyrosine, with the first two being preferable.

Histidine, arginine and tryptophan have an amino group that cancoordinate a transition metal atom. In Histidine, the amino group islocated within a cyclic imidazole ring and hence the lone pair electrons(lpe) of the nitrogen are more available for coordination, as comparedwith aliphatic amines such as in arginine.

In tryptophane, the amino group is located within an imidazole ring thatis conjugated to an aryl ring and hence, its lpe is less available sinceit participates in the it electron system.

Typically, any compound that has an aliphatic or alicyclic amino group(i.e., substituted or unsubstituted) can mimic these amino acidchelating effect, with preference to imidazole-like compounds such asphorphyrin. It will be appreciated that Lysine also has an aliphaticamino group, however, at neutral pH conditions the amino group isprotonized and hence cannot coordinate transition ions.

Cysteine and Methionine have a sulfur-containing group (i.e., —SH and—S—, respectively), which can also coordinate a transition metal atom.Any compound that includes such a group can exert the same chelatingeffect.

Tyrosine, Serine and Threonine have an hydroxyl group, either aromatic(Tyrosine) or aliphatic (Serine and Threonine), which exert a lesspreferable chelating effect, due to possible oxidation of the metal. Anycompound which includes such a group or, alternatively, anoxygen-containing group such as ether (—O—), can exert the samechelating effect.

Methods of producing the compositions of the present invention are wellknown in the art. General metal-insertion protocols are disclosed inSmith, “Porphyrins and Metalloporphyrins, K. M. Smith ed., ElsevierScientific Publishing Co., New York, (1975).

A specific procedure for synthesizing metalloporphyrins in a lowtemperature (i.e., 40)° is disclosed in U.S. Pat. No. 6,420,553.

Briefly, hydrophobic or hydrophilic porphyrin compounds having at leastone porphyrin ring or rings and optionally bearing various substituentgroups can be used as starting materials permitting insertion of atransition metal ion.

The transition metal salts to be complexed with such porphyrin compoundsmay be any of various salts of the transition metals, describedhereinabove, which are able to make at least two formal bonds. The saltof such a transition metals may be any of various inorganic acids, suchas hydrochloric acid, sulfuric acid, phosphoric acid, and variousorganic acids. The transition metal salt is preferably used in a molarexcess over the starting material porphyrin.

While conducting the reaction, the starting material porphyrin and thetransition metal salt are dissolved each in an independent solvent andthe resulting solutions are combined.

The solvent for dissolving the starting material porphyrin is selectedaccording to the hydrophobicity of the porphyrin. A solvent having ahigh solubilizing power for the porphyrin and capable of providing ahomogeneous solution is preferably used. For example, when a hydrophobicporphyrin is reacted, hydrophobic organic solvents are preferably used.Thus, halogenated hydrocarbons, aromatic hydrocarbons, nitriles and thelike may be used.

Alternatively, when the starting material porphyrin is hydrophilic,hydrophilic solvents such as water, alcohols, amines, nitrogeneousheterocyclic compounds are preferably used.

The homogeneous solutions, thus prepared, are combined in the presenceof a basic substance or basic compound. The presence of a basicsubstance can be assured, for example by using a basic substance as thesolvent when it is an amine or a nitrogeneous heterocyclic compound orby adding a basic substance to the combined solution, or even by usingsuch procedures in combination. The relative amount of the solvent andbasic substance can vary.

Examples of basic substances which can be used include, but are notlimited to, nitrogen-containing heterocyclic compounds such as pyridine,methylpyridine, dimethylpyridine, diazines, methyldiazines, pyrazine,ethylpyrazine, pyrimidine, piperazine, morpholine; aliphatic amines suchas diethylamine, ethylenediamine, tert-butylamine, basic resins; andinorganic bases.

The reaction is preferably effected at 40° C., although lowertemperature conditions (i.e., room temperature) can also mediate thereaction.

Once the reaction is complete, the reaction product can be isolated andpurified by various procedures such as chromatography, precipitation,recrystallization and the like.

Alternatively, the compositions of the present invention can becommercially obtained such as from Frontier Scientific PorphyrinProducts (www.porphyrin.com).

In any case, once the composition of the present invention is obtained,it is used to generate antibodies or antibody fragments thereto.

As used herein the term “antibody”, refers to an intact antibodymolecule and the phrase “antibody fragment” refers to a functionalfragment thereof, such as Fab, F(ab′)₂, and Fv that are capable ofbinding to macrophages. These functional antibody fragments are definedas follows: (i) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule, can be produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (ii) Fab′, the fragment ofan antibody molecule that can be obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (iii) (Fab′)₂, the fragment of the antibody that can beobtained by treating whole antibody with the enzyme pepsin withoutsubsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments heldtogether by two disulfide bonds; (iv) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; (v)Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule; and (vi) Peptides coding for asingle complementarity-determining region (CDR).

Methods of generating antibodies (i.e., monoclonal and polyclonal) arewell known in the art. Antibodies may be generated via any one ofseveral methods known in the art, which methods can employ induction ofin vivo production of antibody molecules, screening immunoglobulinlibraries or panels of highly specific binding reagents as disclosed[Orlandi D. R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837, WinterG. et al. (1991) Nature 349:293-299] or generation of monoclonalantibody molecules by continuous cell lines in culture. These includebut are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the Epstein-Bar-Virus (EBV)-hybridoma technique[Kohler G., et al. (1975) Nature 256:495-497, Kozbor D., et al. (1985)J. Immunol. Methods 81:31-42, Cote R. J. et al. (1983) Proc. Natl. Acad.Sci. 80:2026-2030, Cole S. P. et al. (1984) Mol. Cell. Biol.62:109-120].

In cases where the invention compounds are too small to elicit a strongimmunogenic response, such antigens (haptens) can be coupled toantigenically neutral carriers such as keyhole limpet hemocyanin (KLH)or serum albumin [e.g., bovine serum albumine (BSA)] carriers (see U.S.Pat. Nos. 5,189,178 and 5,239,078 and Examples 2 of the Examplessection). Coupling to carrier can be effected using methods well knownin the art; For example, direct coupling to amino groups can be effectedand optionally followed by reduction of imino linkage formed.Alternatively, the carrier can be coupled using condensing agents suchas dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.Linker compounds can also be used to effect the coupling; bothhomobifunctional and heterobifunctional linkers are available fromPierce Chemical Company, Rockford, Ill. The resulting immunogeniccomplex can then be injected into suitable mammalian subjects such asmice, rabbits, and the like. Suitable protocols involve repeatedinjection of the immunogen in the presence of adjuvants according to aschedule which boosts production of antibodies in the serum. The titersof the immune serum can readily be measured using immunoassay procedureswhich are well known in the art.

The antisera obtained can be used directly or monoclonal antibodies maybe obtained as described hereinabove.

Antibody fragments can be obtained using methods well known in the art.(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988, incorporated herein byreference). For example, antibody fragments according to the presentinvention can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli or mammalian cells (e.g. Chinese hamster ovarycell culture or other protein expression systems) of DNA encoding thefragment.

Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778.

CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.

It will be appreciated that for human therapy or diagnostics, humanizedantibodies are preferably used. Humanized forms of non-human (e.g.,murine) antibodies are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will include at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Once antibodies are obtained, they may be tested for metalloproteininhibitory activity. Appropriate assay conditions for metalloproteininhibition activity are described in Knight et al., FEBS Letters296(3):263-266(1992), Cawston et al., Anal. Biochem, 99:340-345 (1979),Cawston et al., Methods in Enzymology 80:771 et seq. (1981); Cawston etal., Biochem. J., 195:159-165 (1981), Weingarten et al., Biochem.Biophys. Res. Comm., 139:1184-1187 (1984) and U.S. Pat. Nos. 4,743,587and 5,240,958.

Using the methodology described hereinabove, the present inventors wereable to produce matrix metalloprotease (MMP) inhibitory antibodies forMMP-2 and MMP-9.

Thus, according to another aspect of the present invention there isprovided an antibody or an antibody fragment, which is capable ofinhibiting the activity of an MMP. The antibody or antibody fragment ofthis aspect of the present invention includes an antigen recognitionregion (antigen binding region) that is capable of binding a structure,which includes a metal ion coordinately bound within a chelator thereof.

As is illustrated in the Examples section which follows, the presentinventors have conclusively shown that such an antibody raised against achelator-metal ion structure present in an MMP is capable ofspecifically and efficiently inhibiting the activity of an active formof such an enzyme.

A matrix metalloprotease refers to an enzyme which degrades connectivetissues and connective tissue components and which includes a catalyticdomain having a chelating sequence, such as the zinc-binding motif setforth in HEXXHXXGXXH and a transition metal ion such as Zinc.

Table 1 below, lists a number of vertebrate MMPs, as well asnon-vertebrate members, which have been identified in sea urchins,Caenorhabditis elegans, soybean, and Arabidopsis thaliana and which canbe used as targets for the potent inhibitors of this aspect of thepresent invention.

TABLE 1 Protein MMP Collagenase 1 MMP-1 Gelatinase A MMP-2 Stromelysin 1MMP-3 Matrilysin MMP-7 Collagenase 2 MMP-8 Gelatinase B MMP-9Stromelysin 2 MMP-10 Stromelysin 3 MMP-11 Macrophage elastase MMP-12Collagenase 3 MMP-13 MT1-MMP MMP-14 MT2-MMP MMP-15 MT3-MMP MMP-16MT4-MMP MMP-17 Collagenase 4 (Xenopus) MMP-18 (No trivial name) MMP-19Enamelysin MMP-20 XMMP MMP-21 CMMP MMP-22 (No trivial name) MMP-23

Since the chelating sequence of MMP includes histidine residues whichare imidazole based ligands as described above, the metal chelatorutilized for generating MMP neutrilizing antibodies according to theteachings of the present invention preferably includes at least twoporphyrin-like molecules, which are imidazole-like structures.

A variety of synthetic porphyrins are known in the art. Examples includebut are not limited to 5,10,15,20-Tetra(4-pyridyl)porphyrin,5,10,15,20-Tetra(4-pyridyl)porphyrin,5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrintetra(p-toluenesulfonate),5,10,15,20-Tetrakis(4-trimethylammoniophenyl)porphyrintetra(p-toluenesulfonate) and5,10,15,20-Tetrakis(pentafluorophenyl)porphyrin. Such porphyrins arecommercially available from Sigma-Aldrich (www.sigmaaldrich.com).

Examples of compositions which can be used to generate the inhibitoryantibodies of this aspect of the present invention include, but are notlimited to, “capped” porphyrins [Almog, J., et al., J. Am Chem.97:226-227 (1975); Almog, J., et al., Tetrahedron 37:3589-3601 (1981);Baldwin, J. E., et al., J Chem. Soc., Dalton Trans. pp. 1739-1746(1984)], “bridged” porphyrins [Battersby, A. R., et al., J. Chem. Soc.,Chem. Comm. pp. 879-891 (1976); Battersby, A. R., et al., TetrahedronLett. 3169-3172 (1978)], “picket fence” porphyrins [Collman, J. P., etal., J Am. Chem. Soc. 95:7868-7870 (1973); Collman, J. P., et al., J.Am. Chem. Soc. 97:1427-1439 (1975)], “pocket” porphyrins [Collman, J.P., et al., Inorg. Chem. 22:1427-1432 (1983)], “basket-handle”porphyrins [Momenteau, M., et al., Nouv. J. Chim. 3:77-99 (1979);Momenteau, M., et al., J. Mol. Catal. 7:315-320 (1980); Momenteau, M.,et al., J. Chem. Soc. Perkins Trans. 1:189-196 (1983)], “gyroscope”porphyrins [Lecas, A., et al., Tetrahedron Lett. pp. 1019-1022 (1985);Boitrel, B., et al., J. Chem. Soc. Chem. Comm. pp. 1820 (1985)],“cyclophane” porphyrins [Diekmann, H., et al., J. Am. Chem. Soc.93:4068-4070 (1971); Traylor, T. G., et al., J. Am. Chem. Soc.107:604-614 (1985)], and “jelly-fish” type porphyrins [Uemori, Y., etal., Inorg. Chem. 28:1690-1694 (1989)].

As is mentioned hereinabove, one specific use for the antibodies of thepresent invention is prevention or treatment of diseases associated withimbalanced or abnormal activity of metalloproteins such asmetalloproteases.

Examples of such disease include, but are not limited to, arthriticdiseases, such as osteoarthritis (OA), rheumatoid arthritis (RA), septicarthritis, soft tissue rheumatism, polychondritis and tendonitis;metastatic tumors, periodontal diseases; corneal ulceration, such asinduced by alkali or other burns, by radiation, by vitamin E or retinoiddeficiency; glomerular diseases, such as proteinuria, dytrophobicepidermolysis bullosa; bone resorption diseases, such as osteoporosis,Paget's disease, hyperparathyroidism and cholesteatoma; birth controlthrough preventing ovulation or implantation; angiogenesis relating totumor growth or to the neovascularization associated with diabeticretinopathy and macular degeneration; coronary thrombosis associatedwith atherosclerotic plaque rupture; pulmonary emphysema, wound healingand HIV infection.

Thus, according to another aspect of the present invention there isprovided a method of inhibiting matrix metalloprotease activity in asubject in need thereof.

Preferred individual subjects according to the present invention aremammals such as canines, felines, ovines, porcines, equines, bovines,humans and the like.

The method includes providing to the subject a therapeutically effectiveamount of the MMP inhibitor of the present invention (i.e., the antibodyor antibody fragments, described hereinabove).

As is further detailed hereinbelow, the MMP inhibitor can be providedvia direct administration (e.g., oral administration or injection) or itcan be expressed from a polynucleotide construct administered to targetcells of the individual.

The MMP inhibitors of the present invention can be provided to anindividual per se, or as part of a pharmaceutical composition where itis mixed with a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the antibody preparation,which is accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media (Mutter et al. (1979).

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer a preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient. Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro assays. For example, a dose can be formulated in animal modelsand such information can be used to more accurately determine usefuldoses in humans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1].

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

As described hereinabove, the antibody inhibitors of the presentinvention can be expressed from a nucleic acid construct.

It will be appreciated that polynucleotides encoding the antibodies ofthe present invention preferably further encode a signal peptide whichallows secretion or trafficking of the antibodies into a subcellular orextracellular localization of interest. For example, when the targetmetalloprotein is an MMP, a secretory signal peptide is preferablyconjugated inframe to the polynucleotide encoding antibody segment.

It will be further appreciated that recombinant single-chain Fv (ScFv)fragments may be preferably expressed because of their considerably lesscomplicated structure as compared to whole antibody molecules. Asdescribed hereinabove ScFvs are proteins consisting of the V_(L) andV_(H) antibody polypeptide chains synthesized as a single chain with thecarboxyl terminus of V_(L) linked by a peptide bridge to the aminoterminus of V_(H) Methods for recombinantly producing these peptides arewell known in the art [see Bird et al., Science 242:423-426 (1988);Huston et al., Proc. Nat'l Acad. Sci. USA 85:5879-5883 (1988); and deKruif et al., J. Mol. Biol. 248:97-105 (1995)]. According to embodimentsof this aspect of the present invention, following immunization with thecompounds of the present invention, splenic, mRNA is harvested from theimmunized animal and used to produce a cDNA library in a bacteriophagewhich displays the ScFv fragments. Phage particles are then screened todetermine those that interact specifically and preferably with theactivated form of the metallop[rotein of interest. ScFv segments arerecovered from these phage particles, and cloned into an expressionconstruct (see U.S. Pat. No. 5,800,814).

The nucleic acid constructs of this aspect of the present invention canbe administered to target cells of the individual subject (i.e., in-vivogene therapy).

Alternatively, the nucleic acid construct is introduced into a suitablecell via an appropriate gene delivery vehicle/method (transfection,transduction, homologous recombination, etc.) and an expression systemas needed and then the modified cells are expanded in culture andreturned to the individual (i.e., ex-vivo gene therapy).

To enable cellular expression of the antibodies or antibody fragments ofthe present invention, the nucleic acid construct of the presentinvention further includes at least one cis acting regulatory element.As used herein, the phrase “cis acting regulatory element” refers to apolynucleotide sequence, preferably a promoter, which binds a transacting regulator and regulates the transcription of a coding sequencelocated downstream thereto.

Any available promoter can be used by the present methodology. In apreferred embodiment of the present invention, the promoter utilized bythe nucleic acid construct of the present invention is active in thespecific cell population transformed. Examples of cell type-specificand/or tissue-specific promoters include promoters such as albumin thatis liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277],lymphoid specific promoters [Calame et al., (1988) Adv. Immunol.43:235-275]; in particular promoters of T-cell receptors [Winoto et al.,(1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983)Cell 33729-740], neuron-specific promoters such as the neurofilamentpromoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477],pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916]or mammary gland-specific promoters such as the milk whey promoter (U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).The nucleic acid construct of the present invention can further includean enhancer, which can be adjacent or distant to the promoter sequenceand can function in up regulating the transcription therefrom.

The constructs of the present methodology preferably further include anappropriate selectable marker and/or an origin of replication.Preferably, the construct utilized is a shuttle vector, which canpropagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for propagation in cells, or integration in a gene and atissue of choice. The construct according to the present invention canbe, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, avirus or an artificial chromosome.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide or antibody froma host cell in which it is placed. Preferably the signal sequence forthis purpose is a mammalian signal sequence. Optionally, the constructmay also include a signal that directs polyadenylation, as well as oneor more restriction sites and a translation termination sequence. By wayof example, such constructs will typically include a 5′ LTR, a tRNAbinding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof. Other vectors can be usedthat are non-viral, such as cationic lipids, polylysine, and dendrimers.

Preferred modes for executing gene therapy protocols are provided inSomia and Verma [(2000) Nature Reviews 1:91-99], Isner (2002) Myocardialgene therapy. Nature 415:234-239; High (2001) Gene therapy: a 2001perspective. Haemophilia 7:23-27; and Hammond and McKirnan (2001)Angiogenic gene therapy for heart disease: a review of animal studiesand clinical trials. 49:561-567.

Because of the ability of the antibodies of the present invention todifferentially recognize the activated form of metalloprotein (seeExamples 5 and 6 of the Examples section), they can be used as potentdiagnostic and prognostic tools, such as by monitoring MMP activity in abiological sample [i.e., any body sample such as blood (serum orplasma), sputum, ascites fluids, pleural effusions, urine, biopsyspecimens, isolated cells and/or cell membrane preparation]. This is ofspecial significance when evaluating the metastatic features of cancercells, wherein imbalanced activation of MMPs facilitate tumor invasion.Likewise, the antibodies of the present invention can be used inmonitoring therapeutic dosage of MMP inhibitors. For such applicationsthe antibodies of the present invention are preferably labeled with eachof any radioactive, fluorescent, biological or enzymatic tags or labelsof standard use in the art. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241.

It will be appreciated that such detection methods can also be used forhigh throughput screening of novel MMPs. Briefly, multiple biologicalsamples can be contacted with the antibodies of the present invention,where activated MMPs can bind thereto. Measures are taken to usebiological samples, which include activated MMPs such as those derivedfrom tumor cell-lines. Typically, a radioactive label is used to reducethe assay volume.

Alternatively, the antibodies of the present invention can be used topurify active metalloenzymes from biological samples.

Numerous protein purification methods are known in the art. For example,the antibodies or antibody fragments of the present invention can beused in affinity chromatography for isolating the metalloenzymes.Columns can be prepared where the antibodies are linked to a solidsubstrate, e.g., particles, such as agarose, Sephadex, and the like, andthe biological sample, such as a cell lysate may be passed through thecolumn, the column washed, followed by increasing concentrations of amild denaturant, whereby the purified metalloenzyme will be released.

The antibodies or fragments thereof generated according to the teachingsof the present invention can be included in a diagnostic or therapeutickit. Antibodies or antibody fragments can be packaged in a one or morecontainers with appropriate buffers and preservatives and used fordiagnosis or for directing therapeutic treatment.

Thus, the antibodies or fragments thereof can be each mixed in a singlecontainer or placed in individual containers. Preferably, the containersinclude a label. Suitable containers include, for example, bottles,vials, syringes, and test tubes. The containers may be formed from avariety of materials such as glass or plastic.

In addition, other additives such as stabilizers, buffers, blockers andthe like may also be added. The antibodies of such kits can also beattached to a solid support, such as beads, array substrate (e.g.,chips) and the like and used for diagnostic purposes. The kit can alsoinclude instructions for determining if the tested subject is sufferingfrom, or is at risk of developing, a condition, disorder, or diseaseassociated with expression of an MMP of interest.

The present invention also provides a novel approach for qualifyingspecificity of antibodies to metal ion and chelators thereof bydetermining conformational changes and/or electronic changes in bindingof the metal ion to the chelator thereof following binding of theantibody.

Determining conformational and electronic changes in binding of themetal ion to the chelator thereof following binding of the antibodyaccording to this aspect of the present invention can be effected byx-ray absorption spectroscopy (XAS) studies. XAS refers to theoscillatory structure in the X-ray absorption coefficient above an X-rayabsorption edge of a target element. This structure represents a uniquesignature of a given material, which depends on the detailed atomicstructure, electronics, and vibrational properties of the material. Acharacteristic XAS spectrum consists of the X-ray Absorption Near EdgeSpectra (XANES) and the Extended X-ray Absorption Fine Structure (EXAFS)regions. The XANES include information about the oxidation state of themetal ion and its geometry and the analysis of the EXAFS region providesthe atomic structure around a given metal ion including the average bonddistances, mean square variation in distance, coordination numbers andthe identity of the atoms around the metal ion.

Typically, the goal of X-ray absorption studies of proteins has been theinvestigation of the local atomic environment, within few nearestneighboring shells, around the element of interest. The X-ray absorptioncross-section measured in the energy range from the absorption edgeenergy of the element through ca. 1000 eV past it provides theinformation about both the structural and electronic properties of theabsorber. EXAFS is a valuable technique for structural elucidation of avariety of metal sites in metalloproteins (Scott see above ref). Thehigh-resolution structural information that can be obtained by XASstudies makes it an advantageous tool to monitor active site zinccoordination and electronics in metal binding proteins such as MMPsduring different stages of the activation and inhibition processes(Kleifeld Supra). It will be appreciated that XAS is the onlyspectroscopic tool available to date that can probe directly theotherwise spectroscopically silent zinc ion. Additional details on EXAFSare provided by Scott, R. A. (1985) Methods in Enzymology, 117.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Preparation of Monoclonal Antibodies Against the MMP-2 ActiveSite

For the production of a specific monoclonal antibody against theelectronic and physical features of the MMP-2 active site synthetichaptens (FIG. 3) with an analogue structure to the MMP-2 active site,i.e., a zinc ion coordinated to three histidines and a water molecule(Kleifeld et al., 2001), were synthesized and further used for miceimmunization.

Material and Experimental Methods

Synthesis of Co-TCPP and Zn-TCPP Haptens—

The tetra-carboxy phenyl porphyrin Co(II) (Co-TCPP) and thetetra-carboxy phenyl porphyrin Zn(II) (Zn-TCPP) haptens were synthesizedas follows: 63 mM of either Co(OAc)₂.4H₂O or Zn(OAc)₂.2H₂O in a solutioncontaining methanol and acetic acid at equal concentrations were addedto 6.3 mM TCPP. The mixtures were heated for 30 min at 60° C. duringwhich the organic solvents evaporated. The residues were dissolved inthe same methanol/acetic acid solution and purified using a silicacolumn [DC Scientific, Ohaio Valee, Kelton, Pa. 19346, USA. See Harada,A. et al, Photochemistry and Photobiology (1999) 70, 298-302]. Purifiedfractions were further evaporated and yielded the Co-TCPP or Zn-TCPPhaptens as brown-purple or purple sediments, respectively. The identityof the sediments was further confirmed using mass spectrometry.

Gelatin Zymography—

For substrate zymography, a purified protein sample was loaded on a 12%SDS-PAGE containing 0.5 mg/ml gelatin. Activity assay was conducted asdescribed elsewhere (Gogly et al., 1998).

Conjugation of Haptens to Carrier Proteins—

The Co-TCPP and Zn-TCPP haptens (4.6 μmole each) were dissolved in 475μl of dimethylformamide (DMF) and activated for 1 hr at room temperature(RT) by 3.2 μmole of carbonyldiimmidazole (CDI, Fluka, 21860,Sigma-Aldrich Israel Ltd. Rehovot, Israel). Activated haptens were addedto cold solutions of 2.5 mg/ml bovine serum albumin (BSA) or of 2.5mg/ml keyhole limpet hemocyanin (KLH), both in 0.1 M NaHCO₃, pH 8.Haptens were stirred for 3 hours at 4° C. and for an additional 1 hourat RT. Hapten conjugates were dialyzed extensively in a solutioncontaining 0.1 M NaHCO₃, pH 8 and 3×PBS at a molar concentration of 0.5mM and diluted to a final concentration of 1 mg/ml and 0.6 mg/ml forCo-TCPP-BSA and Co-TCPP-KLH, respectively, and to a final concentrationof 1 mg/ml for both Zn-TCPP-BSA and Zn-TCPP-KLH. For hapten density (Hd)determination hapten conjugates were diluted (1:50) in phosphatebuffered saline (PBS) and absorbance was measured at 431 nm. Haptendensity per carrier molecule was calculated according to the estimatedmolar concentration.

Immunization of Mice Against the TCPP Haptens—

Mice were immunized by a foot pad injection of 50 μg haptens conjugatedto KLH. Following two boost injections in two weeks intervals mice wereeyebled to assess antibody titer. One month following the second boostinjection a tail vein injection was given (50 μg in PBS) and 3 dayslater the spleen was fused with poly ethylene glycole (PEG) myeloma cellline to produce hybridomas. Hybridomas were cloned into 96-well platesand screened against hapten conjugates by immunoabsorbent ( ) assay(ELISA) essentially as described elsewhere [Engvall E. and Pece A. J.(1978) Scand. J. Immunol. 8, Suppl. 7].

Purification of Monoclonal Antibodies—

Ascites samples were mixed with an equal volume of (NH₄)₂SO₄ andcentrifuged for 20 min at 10,000 rpm at 4° C. The pellet wasre-suspended in PBS, incubated for 1 hour and dialyzed for 4 hours.Monoclonal antibodies were further purified by protein G Sepharosecolumn (Pharmacia, 17-0618-01, Rehovot, Israel) according tomanufacturer's instructions. Purified antibodies were dialyzed again inthe same solution for 4 hours hours and antibody concentration wasdetermined by the absorption at 280 nm.

Experimental Results

The Production of Monoclonal Antibodies Against the Electronic andPhysical Features of MMP-2—

In order to produce specific antibodies that would recognize theelectronic and physical features of the MMP-2 catalytic site synthetichaptens corresponding to the catalytic active site were synthesized. Inthese haptens the cobalt (Co) or zinc (Zn) atoms are linked to thetetra-carboxy phenyl porphyrin molecule (Co-TCPP or Zn-TCPP,respectively) as illustrated in FIG. 3. Prior to mice immunization thehaptens were conjugated to bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH).

Two groups of mice were immunized with Co-TCPP or Zn-TCPP, conjugated toKLH. The serum titer was measured from mice eyebleed 7-10 days followingthe second and third hapten-boost injection. While mice immunized withthe Co-TCPP hapten exhibited a sufficient serum titer of 1:20,000, miceimmunized with the Zn-TCPP hapten exhibited a very low and insufficienttiter (not shown). Three days following the third boost injection thespleen was removed and fused with myeloma cells. One hundred and twentyclones secreting antibodies were obtained.

These results demonstrate that the Co-TCPP hapten, but not the Zn-TCPPhapten, is stable and suitable for antibody production. These resultscould be explained by destabilization of the water-zinc bond of theZn-TCPP hapten in vivo.

Example 2 Identification and Classification of Monoclonal Antibodies

Mononclonal antibodies were identified and classified by their affinityand competition to conjugated haptens.

Materials and Experimental Methods

Affinity of the Monoclonal Antibody to Conjugated Haptens—

Microtiter plates (Maxi Immunoabsorp, Nunc, Rehovot, Israel) were coatedfor 1 hour with 3 μg/ml of Hapten conjugates in PBS, blocked for 1 hourin 1% BSA/PBS and further subjected to 1 hour incubation with variousconcentrations of monoclonal antibodies. Coated plates were then washedthree times in 0.04% Tween-20 (Sigma, rehovot, Israel) in PBS followedby 1 hour incubation with a secondary goat anti mouse peroxidaseconjugated antibody (Jackson ImmunoResearch Laboratories Inc., Tel-Aviv,Israel). Plates were then washed again in PBS and peroxidase reactionswere developed in a solution containing 0.028M citric acid, 0.044 MNa₂HPO₄, 1 mg/ml ABTS (2,2, azino-bis(3-Ethylbentiazoline-6-sulfonicacid) and 30% H₂O₂.

Characterization of Antibody Subclasses—

Monoclonal antibodies (1 μg in 50 μl) were added to hapten-coatedmicrotiter plates and incubated for 1 hour at RT. Plates were thenwashed in PBS containing 0.04% Tween-20 and various types of secondaryperoxidase conjugated antibodies (IgG₁, IgG_(2a), IgG_(2b), IgG_(g3),IgM) were added for 1 hour incubation. Antibody-bound plates were thenwashed in PBS and peroxidase reactions were developed as describedhereinabove.

Experimental Results

Hybridoma Screening—

Isolated hybridomas were grown for one week. Hybridoma conditioned mediawere then analyzed for ability to bind a solid phase bound haptenantigen. Fifty positive hybridomas (out of 120) were further analyzedfor the ability of the antigen to inhibit antibody binding to theimmunizing hapten conjugated to BSA. This analysis resulted in 17hybridomas in which 50% inhibition was achieved in less than 10⁻⁶Mhapten concentration (data not shown).

Antibody Subclasses—

Antibody-secreting hybridomas were classified to subclasses by bindingto IgG₁, IgG_(2a), IgG_(2b), IgG_(g3), or IgM secondary antibodies.Thirteen out of 17 hybridomas were identified as monoclonal antibodiesof them one was IgM and 12 were IgGs (not shown). Four monoclonalhybridomas with the lowest value for 50% inhibition (e.g., lower than10⁻⁷ M) were further subcloned and tested for MMP-2 inhibition. Amongthem the antibody with the best inhibition effect (Co-TCPP13e11) waspropagated as ascites.

These results demonstrate the production of a specific monoclonalantibody targeted at the Co-TCPP hapten.

Example 3 Characterization of MMP-2 Monoclonal Antibody

To further characterize the Co-TCPP monoclonal antibody competitiveassays with hapten competitors and immuno-precipitation assays wereperformed.

Materials and Experimental Methods

Competitive Assay—

Monoclonal antibodies (at 50% binding concentration) were incubated for1 hour with the TCPP, Co-TCPP, Zn-TCPP, Zn-TPP hapten competitors andwere then transferred to hapten-coated microtiter plates as described inExample 2 hereinabove. The dissociation constant required to attain 50%binding was determined.

Immuno Precipitation—

Protein A beads Pharmacia, Rehovot, Israel were washed in PBS accordingto manufacturer instructions. Ten μg of monoclonal antibody in 300 μlPBS were added to ˜30 μl of washed protein A beads and incubated overnight at 4° C. Protein A-bound antibodies were then washed three timesin PBS and further incubated for 3 hours at RT with 1 μg of MMP-2catalytic domain [MegaPharm (Oncogene Research Products), Hod-Hasharon,Israel, Cat. #PF023] continuous shaking. Following incubation themixture was centrifuged for 2 min at 3000 rpm and pellet and supernatantphases were analyzed on a 12% SDS-PAGE gel.

Kinetic Assays Using a Fluorogenic Substrate—

The inhibitory effect of the antibodies of the present invention on thehydrolysis of the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ fluorogenicsubstrate (synthesized in the laboratory of Pof. Fridkin at the WeizmannInstitute) by the MMP-2 catalytic domain protein was measured asdescribed elsewhere (Netzel-Arnett et al., 1991). Briefly, MMP-2 samples(200 nM) were added to the fluorogenic substrate at a concentrationrange of 0.4-50 μM in a final volume of 200 The reaction was performedat 37° C. and enzyme activity was monitored every 8 seconds along 30minutes by measuring the fluorescence intensity (emission at 390 nm,excitation at 340 nm) using the SPECTRAFluor Plus apparatus (TECAN,Pharatop, Tel-Aviv, Israel). The V, was calculated for each substrateconcentration by the linear part of each kinetic curve and the kineticparameters (K_(M) and V_(MAX)) were calculated from the Lineveawer-Burkplot.

Experimental Results

The Affinity of the Monoclonal Antibody to Co-TCPP Hapten—

The Co-TCPP13e11 monoclonal antibody was characterized by its affinityto the immunizing Co-TCPP hapten. Serial dilutions of the purifiedantibody were added to Co-TCPP—coated microtiter plates. The IC₅₀ value,which corresponds to the antibody dilution required for 50% binding, wascalculated from the titration curve (FIG. 4) and was found to be˜20,000.

Competitive Assay Revealed Antibody Specificity Towards the ImmunizingHapten—

To determine whether the antibody binds specifically to the immunizinghapten, the monoclonal antibody was incubated with Co-TCPP, Zn-TCPP,TCPP, and Zn-TPP and titration curves were plotted against haptenconcentrations (FIG. 5). The dissociation constants, i.e., theconcentration which results in 50% binding of the antibody to thehapten, were 9×10⁻⁸ M, 1×10⁻⁵ M, 2×10⁻⁵M, and 3×10⁻⁵M for Co-TCPP,Zn-TCPP, TCPP, and Zn-TPP respectively. These values demonstrate thehigh specificity of the antibody towards the immunizing hapten(Co-TCPP).

Specific inhibition of MMP-2 and MMP-9 activity by the monoclonalantibody—To determine whether Co-TCPP13e11 monoclonal antibody canspecifically inhibit the MMP proteins the hydrolysis of the fluorogenicsubstrate was measured in the presence of the antibody. The kineticparameters of the inhibition assay (i.e., Ki) were 24 μM and 13 μM forthe MMP-2 catalytic domain and MMP-9 full-length enzyme, respectively.Interestingly, at all antibody concentrations the Lineweaver-Burk curvesexhibited a single and common intersect point on the Y axis (FIGS. 6a-b). These results suggest that the monoclonal antibody inhibited theMMP-2 and MMP-9 enzymes in a competitive manner.

The selectivity of the monoclonal antibody towards MMPs was furtherexamined by testing its inhibitory effect on a different zinc—dependentenzyme, the Thermoanaerobacter brockii alcohol dehydrogenase (TbADH).This enzyme catalyses the reversible oxidation of secondary alcohols tothe corresponding ketones using NADP⁺ as a cofactor (Korkhin et al.,1998). The catalytic zinc in TbADH is coordinated to histidine,cysteine, aspartate, and glutamate in a tetraheder configuration.Incubation of the monoclonal antibody with TbADH did not affect theenzyme activity (data not shown).

These results further suggest that the monoclonal antibody recognizesthe exposed imidazole-based histidine protein residues, which arecoordinated to the catalytic zinc ion in MMP's (Morgunova et al., 1999;Kleifeld et al., 2001) and not necessarily the zinc ion itself.

Immuno Precipitation (IP) Analyses with the Monoclonal Antibody RevealedHigh Specificity Towards MMP-2 Catalytic Domain—

To further verify that the monoclonal antibody inhibited the activity ofthe various MMPs by directly interacting with their catalytic site, aseries of immuno-precipitation (IP)—based experiments were conducted.Briefly, the monoclonal antibody was associated with protein A beads andthe MMP-2 catalytic domain enzyme was added for three hours incubation.Following centrifugation the beads-containing phase (i.e., the pellet)and the beads-free phase (i.e., the soluble) were analysed on anSDS-PAGE gel. When the MMP-2 catalytic domain was incubated withantibody-free protein A beads the MMP-2 was contained within the solublephase (FIG. 7 a, lane 1) but not the pellet phase (FIG. 7 a, lane 2). Inaddition, when the monoclonal antibody was incubated with protein Abeads the antibody was included in the pellet phase (FIG. 10 a, lane 4)but not the soluble phase (FIG. 10 a, lane 3). These results ruled outthe possibility of a direct association between the MMP-2 enzyme andprotein A beads. On the other hand, the MMP-2 enzyme was found in thepellet phase when incubated with pre-associated antibody-protein A beads(FIG. 7 a, lanes 5 and 6). To rule out the possibility that the enzymeand antibody associated to form aggregates directly they wereco-incubated in the absence of protein A beads. As shown in FIG. 7 a(lane 9) the soluble phase contained both the antibody and the enzyme.To further study the specificity of the antibody to MMP-2, thepre-associated antibody-bound beads were incubated with the non-relevantRNA-helicase Dead Box Protein A (DbpA). No specific binding was observedin the pellet phase (FIG. 7 a, lanes 7 and 8) indicating lack ofspecific interaction between the antibody and DbpA. Similar results wereobtained with other non-relevant proteins (e.g., the C-terminalcytoplasmic domain of gliotactin, data not shown).

Altogether, these results demonstrate that the monoclonal antibody bindsthe MMP-2 catalytic domain in a highly specific way and form a stablecomplex.

The Zinc Ion is Necessary for Antibody Binding—

The zinc ion of the MMP-2 catalytic domain was chelated using theortho-phenanthroline [Maret W., Makinen, M. W., JBC, 266, 20636-44(1991)] and the enzyme was further incubated with pre-associatedantibody-protein A beads. As shown in FIG. 7 b (lanes 1 and 2) themononclonal antibody did not bind the zinc-free MMP-2 catalytic domain.These results demonrate that the zinc ion in the enzyme is critical forantibody binding. In addition, incubation of the antibody-MMP-2 complexwith the hydroxamate-based high affinity MMP inhibitor, GM-6001 (Bendecket al., 1996), did not affect the integrity of the complex (FIG. 7 c).These results further suggest that the antibody forms a tight, andirreversible complex with the active site of the MMP-2 enzyme.

Altogether, these results demonstrate that the Co-TCPP monoclonalantibody can specifically inhibit the MMP-2 active site. Moreover, theseresults suggest that the antibody initially recognizes theimidazole-based histidine zinc-ligands within the MMP catalytic site,and then binds to the catalytic metal atom.

Example 4 X-Ray Absorption Spectroscopy (XAS) Studies

XAS refers to the oscillatory structure in the X-ray absorptioncoefficient above an X-ray absorption edge of a target element. Thisstructure represents a unique signature of a given material, whichdepends on the detailed atomic structure, electronics, and vibrationalproperties of the material. A characteristic XAS spectrum consists ofthe X-ray Absorption Near Edge Spectra (XANES) and the Extended X-rayAbsorption Fine Structure (EXAFS) regions. The XANES include informationabout the oxidation state of the metal ion and its geometry and theanalysis of the EXAFS region provides the atomic structure around agiven metal ion including the average bond distances, mean squarevariation in distance, coordination numbers and the identity of theatoms around the metal ion.

Materials and Experimental Methods

Sample Preparation for XAS Studies—

Pro-MMP-2 (Fridman et al., 1992; obtained from Prof. R. Fridman, WayneState University, Detroit, USA) was activated by 4-aminophenylmercuricacetate (APMA) as described elsewhere (Kleifeld et al., 2001). Theenzyme and the monoclonal antibody were concentrated by ultrafiltrationusing a Millipore Centricon-10 device (Bedford, Mass., USA) to a finalconcentration of 0.1 mM and 0.2 mM, respectively. Samples were loadedinto copper sample holders (10×5×0.5 mm made in the technical serviceunit at the Weizmann Institute) covered with Mylar tape and were frozenimmediately in liquid nitrogen. Frozen samples were mounted inside aDisplex closed-cycle helium cryostat (Brookhaven Laboratory, New-York,USA).

XAS Data Collection, Processing and Analysis—

XAS studies were performed as described elsewhere (Kleifeld et al.,2001). Briefly, the incident beam intensity, I₀, was recorded using anionization chamber (Brookhaven Laboratory, New-York, USA) and thefluorescence intensity, I_(F), was recorded using a 13-element germaniumdetector (Crambera, Calif., USA).

EXAFS Fitting—

Model data for the fitting procedure were constructed by extracting thecatalytic zinc site coordinates (in a radius of 5 Å) from the structureof a small molecule, WABKIT, that contains a zinc ion coordinated tothree histidines, one oxygen and one sulfur, obtained from the ProteinData Bank (PDB, http://www.rcsb.org/pdb/). The theoretical photoelectronscattering amplitudes and phase shifts were calculated using thecomputer code FEFF7 (Rehr et al., 1991; Zabinsky et al., 1995). Thetheoretical XAFS signal was fitted to the experimental data using thenonlinear least squares fitting method, implemented in the programFEFFIT (Stern et al., 1995).

Experimental Results

The Structure of the MMP-2 Catalytic Site is Changed Upon Binding ofMonoclonal Antibodies—

The local structure of the catalytic zinc ion in the recombinant,full-length human MMP-2 enzyme in its latent (i.e., pro-enzyme), active,and monoclonal antibody-inhibited states were studied using XAS. Thepro-enzyme edge spectrum displayed three distinct peaks at 9668, 9713and 9738 electron volt (eV) and a typical cleft at 9725 eV. Activationof MMP-2 resulted in a slight increase in peak intensity at 9668 eV. Onthe other hand, the inhibition of MMP-2 by the monoclonal antibodyresulted in a higher peak at 9668 eV, an additional peak at 9680 eV(FIG. 8 a, arrow 1) and the absence of evident cleft at 9730 eV (FIG. 8a, arrow 2). In addition, a distinct shift of 0.86 eV to higher energyis observed between the active and the inhibited enzyme (FIG. 8 b).These spectral differences in XANES demonstrate local conformationalchanges in the metal atom of the active site of MMP-2 upon binding ofthe antibody.

Fitting Analysis of MMP-2 Active Site—

In order to further elucidate the conformational changes occurring inthe active site following antibody binding an EXAFS fitting analysis wasperformed. FIG. 9 demonstrates the fitting results of the experimentaldata to theory. Data are presented as the radial distribution from themain absorber (the zinc ion) and the nearest coordination shells. TheF.T. magnitude indicates the peak intensities in arbitrary numbers.These results are consistent with the penta coordination of the zinc ionwith three Zn—N at 2.07 Å, one Zn—S at 2.49 Å, and one Zn—O or Zn—N at1.96 Å and further demonstrate that the antibody binds directly to thecatalytic zinc ion via one Zn—S and one Zn—O or Zn—N ligands.

Altogether these results demonstrate that the monoclonal antibody bindsthe catalytic zinc ion.

Example 5 Complimentary Assays Support the EXAFS Results

To determine whether the antibody can be removed from the MMP-2catalytic site by the cleavage of the Zn—S bond (Van Wart andBirkedal-Hansen, 1990) the antibody—MMP-2 complex was incubated withAPMA.

Experimental Results

The Antibody-MMP-2 Association is Relatively Stable—

The antibody-MMP-2 complex was subjected to APMA treatment as describedelsewhere (Kleifeld et al., 2001) and the soluble and pellet phases werefurther analyzed by IP on an SDS-PAGE gel. As shown in FIG. 10, only asmall portion of the MMP-2 enzyme was released from the antibody-enzymecomplex.

These results demonstrate a relatively low accessibility of the APMAreagent to the catalytic site of the enzyme due to antibody shielding.

Example 6 Anti MMP-2 Monoclonal Antibody Blocks Pericellular Proteolysisof Highly Invasive Fibrocarcinoma Cells

The ability of anti-MMP2 monoclonal antibody of the present invention toinhibit pericellular proteolytic activity generated by highly invasivefibrocarcoma cancer cells (HT1080), was assayed by in situ zymographyassay.

Materials and Experimental Procedures

Cells, Buffers and Growth Conditions—

HT1080 cells were obtained from the American type culture collection(ATCC, Catalog No. CRL-12011). Cells were grown in Dulbecco's modifiedEagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodiumbicarbonate and 4.5 g/L glucose and supplemented with 0.1 mMnonessential amino acids, 90%; fetal bovine serum, 10% at 37° C.

In Situ Zymography Assay—

HT1080 were embedded in fully conjugated fluorescent matrigel andin-situ zymography assay was effected essentially as described by Wanget al. Comp Hepatol. 2004 Jan. 14; 3 Suppl 1:S20; and Nakada Am JPathol. 1999 February; 154(2):417-28.

Experimental Results

HT1080 cells serve as a good model for matrix invasion since theycontain MT1-MMPs MMP-2, MMP-9, and TIMPS. Cells were incubated in thepresence (FIG. 11 b) or absence (FIG. 11 a) of anti MMP-2 monoclonalantibody described above DAPI stained and subjected to an in situzymography assay. As is clearly shown in FIGS. 11 a-b, in the absence ofanti MMP2 antibody (FIG. 11 a), MMP pericellular proteolysis appeared asgreen intensity around the cell membrane. However upon addition of theantibody, this pattern disappeared, demonstrating that the mAb inhibitsthe proteolytic activity of MMPs generated from these highly invasivecancer cells with a Ki of 1 μM. These results therefore suggest that themAb binds and inhibits active MMPs within the pericellular space.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCE LIST Additional References are Cited in the Text

-   1. Nagase, H. and Woessner, J. F. Jr. (1999). Matrix    metalloproteiases.Minireview. J. Biol. Chem. 274: 21491-21494.-   2. Bode, W., Fernandez-Catalan, C., Nagase, H., and Maskos, K.    (1999). Endoproteinase-protein inhibitor interactions. APMIS 107,    3-10.-   3. Bode, W., Fernandez-Catalan, C., Tschesche, H., Grams, F.,    Nagase, H., and Maskos, K. (1999). Structural properties of matrix    metalloproteinases. Cell. Mol. Life. Sci. 55, 639-652.-   4. Borkakoti, N. (1998). Matrix metalloproteases: variations on a    theme. Prog. Biophys. Mol. Biol. 70, 73-94.-   5. Brown, S., Bernardo, M. M., Li, Z. H., Kotra, L. P., Tanaka, Y.,    Fridman, R., and Mobashery, S. (2000). Potent and Selective    Mechanism-Based Inhibition of Gelatinases. J. Am. Chem. Soc., 122,    6799-6800.-   6. Fridman, R., Fuerst, T. R., Bird, R. E., Hoyhtya, M., Oelkuct,    M., Kraus, S., Komarek, D., Liotta, L. A., Berman, M. L., and    Stetler-Stevenson, W. G. (1992). Domain structure of human 72-kDa    gelatinase/type IV collagenase. Characterization of proteolytic    activity and identification of the tissue inhibitor of    metalloproteinase-2 (TIMP-2) binding regions. J Biol. Chem. 267,    15398-405.-   7. Gogly, B., Groult, N., Hornebeck, W., Godeau, G., and Pellat, B.    (1998). Collagen Zymography as a Sensitive and Specific Technique    for the Determination of Subpicogram Levels of Interstitial    Collagenase. Anal. Biochem. 255, 211-216.-   8. Gomez, D. E., Alonso, D. F., Yoshiji, H., and Thorgeirsson, U. P.    (1997). Tissue inhibitors of metalloproteinases: structure,    regulation and biological functions. Eur. J. Cell. Biol. 74,    111-122.-   9. Henriet, P., Blavier, L., and Declerck, Y. A. (1999). Tissue    inhibitors of metalloproteinases (TIMP) in invasion and    proliferation. APMIS 107, 111-119.-   10. Kleifeld, O., Kotra, L. P., Gervasi, D. C., Brown, S.,    Bernardo, M. M., Fridman, R., Mobashery, S., and Sagi, I. (2001).    X-ray Absorption Studies of Human Matrix Metalloproteinase-2 (MMP-2)    Bound to a Highly Selective Mechanism-based Inhibitor. Comparison    with the latent and active forms of the enzyme. J. Biol. Chem. 276,    17125-17131.-   11. Korkhin, Y., Kalb(Gilboa), A. J., Peretz, M., Bogin, O.,    Burstein, Y., and Frolow, F. (1998). NADP-dependent Bacterial    Alcohol Dehydrogenases: Crystal Structure, Cofactor-binding and    Cofactor Specificity of the ADHs of Clostridium beijerinckii and    Thermoanaerobacter brockii. J. Mol. Biol. 278, 967-981.-   12. Morgunova, E., Tuuttila, A., Bergmann, U., Isupov, M.,    Lindqvist, Y., Schneider, G., and Tryggvason, K. (1999). Structure    of Human Pro-Matrix Metalloproteinase-2: Activation Mechanism    Revealed. Science 284, 1667-1670.-   13. Bode, W., Fernandez-Catalan, C., Tschesche, H., Grams, F.,    Nagase, H., and Maskos, K. (1999). Structural properties of matrix    metalloproteinases. Cell. Mol. Life. Sci. 55, 639-652.-   14. Netzel-Arnett, S., Mallya, S. K., Nagase, H., Birkedal-Hansen,    H., and Van Wart, H. E. (1991). Continuously recording fluorescent    assays optimized for five human matrix metalloproteinases. Anal.    Biochem. 195, 86-92.-   15. Rehr, J. J., Mustre de leon, J., Zabinsky, S. I., and    Albers, R. C. (1991). J. Am. Chem. Soc. USA 113, 5135-5138.-   16. Reponen, P., Sahlberg, C., Huhtala, P., Hurskainen, T.,    Thesleff, I., and Tryggvason, K. (1992). Molecular cloning of murine    72-kDa type IV collagenase and its expression during mouse    development. J. Biol. Chem. 267, 7856-7862.-   17. Stern, E. A., Newville, M., Ravel, B., Yacoby, Y., and    Haskel, D. (1995). The UWXAFS analysis package: philosophy and    details. Physica B 208/209, 117-122.-   18. Van Wart, H., and Birkedal-Hansen, H. (1990). The Cysteine    Switch: A Principle of Regulation of Metalloproteinase Activity with    Potential Applicability to the Entire Matrix Metalloproteinase Gene    Family. Proc. Natl. Acad. Sci. USA 87, 5578-5582.-   19. Will, H., Atkinson, S. J., Butler, G. S., Smith, B., and    Murphy G. (1996). The soluble catalytic domain of membrane type 1    matrix metalloproteinase cleaves the propeptide of progelatinase A    and initiates autoproteolytic activation. J. Biol. Chem. 271,    17119-17123.-   20. Zabinsky, S. I., Rehr, J. J., Ankudinov, A., Albers, R. C., and    Eller, M. J. (1995). Multiple-scattering calculations of    x-ray-absorption spectra. Phys. Rev. B 52, 2995-3009.

What is claimed is:
 1. A method of treating a disease associated withabnormal activity of a matrix metalloprotease in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of an antibody comprising an antigen recognition regionwhich binds a transition metal ion-bound chelator, wherein the antibodyinhibits an enzymatic activity of the matrix metalloprotease and whereinsaid antigen recognition region binds a pro-enzyme form of said matrixmetalloprotease with a lower affinity than an active form of said matrixmetalloprotease, thereby treating the disease.
 2. The method of claim 1,wherein said transition metal ion is selected from the group consistingof Vanadium, Selenium, Molybdenum, Cobalt, Zinc, Copper, Iron, Gallium,Bismuth, Aluminum, Gold, Platinum, Manganese, Chronium, Silver,Antimony, Thalium, Cadmium, Nickel, Mercury and Lead.
 3. The method ofclaim 1, wherein said chelator is a polyamine.
 4. The method of claim 3,wherein said polyamine is at least two histidine molecules.
 5. Themethod of claim 3, wherein said polyamine is selected from the groupconsisting of ethylene diamine, cyclam, porphyrin, diethylenetriamine,triethylenetetramine, triethylenediamine, tetraethylenepentamine,aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine,captopril, penicilamine, N,N′-bis(3-aminopropyl)-1,3-propanediamine,N,N′-Bis-(2-animoethyl)-1,3-propanediamine,1,7-dioxa-4,10-diazacyclododecane, 1,4,8,11-tetraazacyclotetradecane-5,7-dione, 1,4,7-triazacyclononane,1-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and1,4,7,10-tetraazacyclododecane.
 6. The method of claim 5, wherein saidpolyamine is porphyrin.
 7. The method of claim 6, wherein saidtransition metal ion-bound chelator is Cobalt(II)-tetra-carbozyl phenylporphyrin (Co-TCPP) or Zinc (II)-tetra-carboxy phenyl porphyrin(Zn-TCPP).
 8. The method of claim 1, wherein said antigen recognitionregion binds to said chelator in an absence of said transition metal ionwith a lower affinity than to said transition metal ion bound chelator.9. The method of claim 1, wherein the antibody has a dissociationconstant for said Co-TCPP of no greater than 9×10⁻⁸ M.
 10. The method ofclaim 1, wherein said matrix metalloprotease is selected from the groupconsisting of neutrophil collagenase, collagenase-3, gelatinase A,gelatinase B, stromelysins-2 and 3, matrilysin, macrophage elastase;membrane-type MMPs, agrrecanase, tumor necrosis factor convertingenzyme, cytokine convertases, adhesion molecule shedding enzymes,endothelin converting enzyme, angiotensin converting enzyme, neutralendopeptidase, FTSH—bacterial metalloprotease, metallo-lactamase(carbapenases), bacterial toxins and ras farnesyl protein transferaseand carbonic anhydrase.
 11. The method of claim 10, wherein said matrixmetalloprotease is gelatinase A.
 12. The method of claim 11, wherein theantibody is capable of inhibiting 50% of an activity of said gelatinaseA at a concentration less than 10⁻⁷M.
 13. The method of claim 1, whereinsaid disease is selected from the group consisting of cancer,osteoarthritis (OA), rheumatoid arthritis (RA), septic arthritis, softtissue rheumatism, polychondritis, tendonitis, periodontal disease,corneal ulceration, proteinuria, dytrophobic epidermolysis bullosa,osteoporosis, Paget's disease, hyperparathyroidism, cholesteatoma;diabetic retinopathy, macular degeneration; coronary thrombosisassociated with atherosclerotic plaque rupture; pulmonary emphysema,wound healing and HIV infection.
 14. The method of claim 1, wherein saidcancer is a metastasized cancer.